Identification of a novel "almost neutral" micro-opioid receptor antagonist in CHO cells expressing the cloned human mu-opioid receptor

Abstract

The basal (constitutive) activity of G protein-coupled receptors allows for the measurement of inverse agonist activity. Some competitive antagonists turn into inverse agonists under conditions where receptors are constitutively active. In contrast, neutral antagonists have no inverse agonist activity, and they block both agonist and inverse agonist activity. The mu-opioid receptor (MOR) demonstrates detectable constitutive activity only after a state of dependence is produced by chronic treatment with a MOR agonist. We therefore sought to identify novel MOR inverse agonists and novel neutral MOR antagonists in both untreated and agonist-treated MOR cells. CHO cells expressing the cloned human mu receptor (hMOR-CHO cells) were incubated for 20 h with medium (control) or 10 microM (2S,4aR,6aR,7R,9S,10aS,10bR)-9-(benzoyloxy)-2-(3-furanyl)dodecahydro-6a,10b-dimethyl-4,10-dioxo-2H-naphtho-[2,1-c]pyran-7-carboxylic acid methyl ester (herkinorin, HERK). HERK treatment generates a high degree of basal signaling and enhances the ability to detect inverse agonists. [(35)S]-GTP-gamma-S assays were conducted using established methods. We screened 21 MOR "antagonists" using membranes prepared from HERK-treated hMOR-CHO cells. All antagonists, including CTAP and 6beta-naltrexol, were inverse agonists. However, LTC-274 ((-)-3-cyclopropylmethyl-2,3,4,4alpha,5,6,7,7alpha-octahydro-1H-benzofuro[3,2-e]isoquinolin-9-ol)) showed the lowest efficacy as an inverse agonist, and, at concentrations less than 5 nM, had minimal effects on basal [(35)S]-GTP-gamma-S binding. Other efforts in this study identified KC-2-009 ((+)-3-((1R,5S)-2-((Z)-3-phenylallyl)-2-azabicyclo[3.3.1]nonan-5-yl)phenol hydrochloride) as an inverse agonist at untreated MOR cells. In HERK-treated cells, KC-2-009 had the highest efficacy as an inverse agonist. In summary, we identified a novel and selective MOR inverse agonist (KC-2-009) and a novel MOR antagonist (LTC-274) that shows the least inverse agonist activity among 21 MOR antagonists. LTC-274 is a promising lead compound for developing a true MOR neutral antagonist.

Figure 1

Effect of DAMGO, HERK and LTC-274 in control (Panel A) and HERK-treated cells (Panel B). Each value is the mean±SD (n=3). The EC₅₀ and E_MAX values are reported in the Results section.

Figure 2

Representative “shift experiments” in membranes prepared from HERK-treated cells with an agonist (dynorphin A(1-13), Panel A) and an inverse agonist (CTAP, Panel B). Each value is the mean±SD (n=4 for Panel A, n=3 for Panel B). The Ke values are reported in Table 3.

Figure 3

Ke value of LTC-274 for agonists and inverse agonists. The Ke values of LTC-274 for agonists and inverse agonists (Table 2) were pooled for statistical analysis. *p<0.01 when compared to agonists (unpaired Student’s t-test).

Figure 4

Effect of pretreatment drug on the efficacy of KC-2-009. Cells were treated for 20 hr in the absence and presence of 10 μM HERK, 10 μM DAMGO, 10 μM gedunin, and 1 μM fentanyl. KC-2-009 dose-response curves (10 data points each) were then generated as described in methods. The inset plots the E_MAX values. *p<0.05 (ANOVA, Dunnett’s Multiple Comparison Test) when compared to control E_MAX. Each value is the mean±SD (n=3).

Figure 5

Opioid antagonist activity of LTC-274 in the 55°C tail flick assay. Mice were pretreated at t = -10 min with vehicle or doses of s.c. LTC-274. At t = 0 min, all mice received an i.c.v. injection of morphine (10 nmol). LTC-274 pretreatment produced dose-related antagonism of the morphine antinociceptive effects at t = 10 and 20 min (times corresponding to agonist peak effect).